【作者】 薛忠林；

【导师】 靳安民；

【作者基本信息】 南方医科大学 ， 骨外科学， 2009， 博士

【摘要】 背景磷酸钙骨水泥（CPC）具有良好的骨传导性和骨替代作用,可以达到原位塑形而与局部缺损相匹配的性能,因此被视为填充骨缺损的良好骨替代材料之一。但目前应用的CPC仍存在一些不足之处,如较低的力学强度,体内难以降解,不可注射等等。由于大孔骨水泥（孔径在100μm以上）能为新骨组织的快速长入提供有利的条件,而且一旦新骨长入,其植入部位的强度便会大大提高。因此从理论上来说,将大孔骨水泥植入体内,在新骨长入以后便可以获得较理想的强度。但是在新骨长入以前,骨水泥的多孔结构又会导致本来就难以承重的骨水泥强度进一步降低,难以维持局部的力学性能和稳定性。所以,提高早期的力学强度对改善大孔骨水泥的性能十分重要。本课题旨在通过将聚乳酸羟基乙酸共聚物（PLGA）微球与骨水泥复合,制备一种早期无孔,晚期通过PLGA微球降解成孔的骨水泥,以达到早期提供强度,后期利于骨组织长入的双重作用。此外,课题组还将这一新型骨水泥作为药物载体,用多柔比星的PLGA微球代替空白微球,试图通过体内外研究证实这种骨水泥的载药和释药性能,降解特点,成骨和抗肿瘤作用等等。目的研究包裹空白微球骨水泥的生物相容性和包裹多柔比星微球骨水泥的力学强度、体外降解和释药特点,观察包裹多柔比星微球骨水泥的成骨作用和抗肿瘤性能。为包裹多柔比星微球骨水泥用于肿瘤切除术后骨缺损修复和抗肿瘤的可行性提供实验依据。方法1包裹PLGA微球骨水泥的生物相容性（1）骨水泥粉末的制备按化学沉淀法合成磷酸钙晶体（PCCP）,将PCCP和无水二磷酸盐（DCPA）按1:1配置而成。（2）微球的制备采用复乳溶剂挥发法制备微球。（3）纯骨水泥的制备以15%的柠檬酸钠作为水相,将CPC按体积重量比0.4 ml·g^-1加入柠檬酸钠溶液并均匀搅拌。然后在特制模具中制备成柱状骨水泥。（4）包裹微球骨水泥的制备将微球与CPC粉末以3:7的比例均匀混合,制备成柱状包裹药物微球骨水泥。（5）实验分组按研究对象不同,将细胞相容性实验分为3组:空白对照组;CPC组,即纯骨水泥组;CPC/M组,即包裹有微球的骨水泥。体内生物相容性研究中又将CPC组和CPC/M组各分为3个浓度或剂量亚组。（6）材料和浸提液的制备浸提液的制备:材料称重,密封后经⁶⁰Co灭菌,以每1g样品加入10ml细胞培养液,在37℃,CO₂体积分数为5%的无菌条件下浸提72h作为浸提液。（7）细胞生长曲线将基质干细胞细胞悬液,接种96孔板,每孔200μL,置于37℃±2℃,CO₂体积分数为5%的培养箱中开放培养24h,使细胞贴壁。弃去培养液,加入等量的浸提液,再放入37℃±2℃,CO₂体积分数为5%的培养箱内分别培养2、4、6、8、10天。在各观察期加入CCK-8试剂,在免疫酶标仪上以450nm波长测定吸光度值。（8）光镜和电镜观察在每次进行细胞增殖实验前,无菌操作下取出培养板,观察浸提液和材料中各孔细胞的生长状态。对材料组和浸提液组的细胞生长状态进行观察、分析。将人骨髓基质干细胞接种于材料上,行扫描电镜观察。（9）急性毒性实验取昆明种小鼠30只,分为生理盐水组（NS）、CPC组和CPC/M组,分别行尾静脉注射生理盐水和2种浸提液原液,每只注射量1ml。注射后24 h、72h和120h称量小鼠体重并观察反应。（10）溶血试验将不同浓度的材料生理盐水浸提液(0.1g·ml^-1、0.05g·ml^-1、0.01 g·ml^-1),生理盐水（阴性对照）和双蒸水（阳性对照）,每组各5份,每管2 ml。再在每管分别加入2%新鲜健康人血悬液2 ml。在分光光度计540nm处测定各样本光密度,计算溶血率。（11）微核试验将80只小鼠随机分成8组:2种材料的低（1ml）、中（2ml）、高（4ml）剂量组,阴性对照组（生理盐水）和阳性对照组（环磷酰胺40mg/kg）。用30h给受试物法。取股骨常规涂片,涂片自然干燥后固定,Giemsa染色10～15min。在油镜下观察。每只动物计数1000个嗜多染红细胞,观察含有微核的嗜多染红细胞数,每组以微核总数占5000个细胞中的比例计算。（12）热原试验健康新西兰白兔6只,间隔1h测量肛温3次,取平均值,确定为正常体温。自耳缘静脉缓慢注入无菌水泥浸提液,5 ml/只,注射后1,2,3 h测定家兔体温3次,与正常体温相比,确定其升高度数。2包裹多柔比星PLGA微球骨水泥的制备和表征（1）实验分组按研究对象不同,将实验分为3组:A组（CPC组）,即材料中只有骨水泥,不含药物和药物微球;B组（CPC/D组）,即含有多柔比星（DOX）药物的骨水泥;C组（CPC/M/D组）,即包裹有DOX药物PLGA微球的骨水泥。（2）电子显微镜观察将微球、纯骨水泥、载药骨水泥、载微球骨水泥材料样本行真空镀膜仪喷金,用扫描电镜在不同放大倍数下观察样本结构特征,测量微球的粒径。（3）X线衍射法将骨水泥粉末与柠檬酸溶液按固液比为1:0.4（g:ml）混合后置于37℃,100%湿度下4周,然后用丙酮浸泡1h,真空干燥,采用X线衍射（XRD）分析仪测试样品的化学成分。（4）骨水泥的抗稀散性按体积重量比0.4 ml·g^-1在骨水泥粉末中加入柠檬酸钠溶液并均匀搅拌,充分混合1min后制成球状,迅速投入到生理盐水（37℃）中静置24h,通过肉眼观察表面稀散情况进行评价。（5）凝结时间分别对3组骨水泥的在25℃和37℃下凝结时间进行测定。用特定装置对初凝时间和终凝时间分别进行测量。每个凝结时间测量6个标本,取平均值。（6）可注射性用带有1.6mm内径针头的,针筒内径为14.5mm的注射器来测量3组骨水泥的可注射性能。在将骨水泥粉末与柠檬酸钠混匀搅拌1min后,立即将其倒入针筒中。在注射器内芯的上方垂直加5kg的压力2min。每组重复6次,记录注射前后的骨水泥的针筒中的骨水泥的质量并计算可注射性。（7）孔隙率的测定根据Archimedean原理,根据排水（乙醇）法来粗略测量样品的孔隙率。（8）抗压强度将试件置于万能生物力学试验机上进行最大抗压强度的测定,施加载荷速度为0.1 mm·min^-1,记录标本样条的最大抗压强度和断裂强度。3包裹多柔比星PLGA微球骨水泥的体外释药和降解（1）载药率与包封率的测定准确称取冷冻干燥的微球5mg溶于二甲亚砜中,超声振荡至微球完全溶解,将微球溶液用超速离心机离心2h,取上部溶液,用紫外分光光度计测定试样在480nm处的吸光度。计算出各自DOX的含量。（2）体外药物释药特性取48个小瓶,每个瓶内放置100mg微球,加入5ml的0.1mol·L^-1 PH7.4的SBF液,37℃下浸泡24小时,随机取6个小瓶的微球,按浸泡后第1、3、5、7、10、14、21、28天留取浸出液,检测各时间点浸出液在480nm处的吸光度,计算微球的释药量。（3）CPC/D和CPC/M/D的体外释药特性从已制备好的2组含有药物成份的骨水泥中各随机抽取2块骨水泥试样,分别置于盛有SBF的烧杯中,测量浸提液在不同时间点的480nm处吸光度。记录每个时间点测得的样本吸光度,并计算出其释药量。（4）载药微球的体外降解精密称取30等份,质量均为100mg的DOX微球和空白微球,分别加入SBF液浸泡,计录不同时间点上微球的重量,计算微球的重量丢失。（5）CPC、CPC/D和CPC/M/D的降解每组各取35个已制备好的干燥骨水泥标本,称重后加入100 ml的SBF中浸泡。每次从各组取出6块标本分别称重,计算骨水泥样本重量丢失;另外6块用于测量其屈服应力并进行电镜观察。4包裹多柔比星PLGA微球骨水泥的体内成骨作用（1）实验动物和分组2周龄新西兰大白兔40只,随机配成20对,将每对动物的4个桡骨依次分成4组:空白组、CPC组、CPC/D组、CPC/M/D组。（2）动物模型的制备实验模型的制备:用3%戊巴比妥钠（1ml/g）耳缘静脉麻醉,然后制备双侧1.5 cm长的骨膜-桡骨缺损。对3个材料组的桡骨行材料植入,对空白组的桡骨只造成缺损模型,但不植入材料。（3）大体观察术后观察动物饮食,活动情况,以及伤口有无肿胀、分泌物出现。取材后观察成骨及材料降解情况。（4）X线检查术后2、4、8、16周各取5对动物拍摄正位X射线片,观察各组动物骨缺损区的骨痂生长、材料降解及骨连接情况。（5）生物力学分别于术后2、4、8、16周随机从3组材料组中取5对兔子处死,切取完整桡骨标本,剔净骨膜及软组织后,测量各组标本弯曲强度,取各组标本的平均值进行统计学分析。（6）骨水泥成分X光衍射图谱确定术后第4周时各组骨残余骨水泥的成分。（7）标本的病理学观察术后16周时分别处死相应组的大白兔,切除双侧桡骨,固定,脱钙,脱水,透明,石腊包埋后切片,烘干后脱蜡,常规HE染色,并在光学显微镜下观察骨水泥在骨内的代谢情况。5包裹载多柔比星PLGA微球骨水泥的抗肿瘤作用（1）肿瘤细胞培养将SaoS-2细胞悬液转移至培养瓶中,37℃培养。（2）材料浸提液的制备随机取3块CPC/M/D样本称重,在37℃,CO₂体积分数为5%的无菌条件下浸提72h分别记作不同浓度的浸提液。（3）肿瘤细胞增殖抑制试验将细胞密度为1×10⁶个/ml的SaoS-2细胞悬液,接种培养5天。每天观察细胞的生长情况并摄片后,弃去培养皿内的浸提液和培养液,加入10μL/孔的CCK-8试剂,在免疫酶标仪上测定其吸光度值。（4）骨肉瘤裸鼠模型的建立用生理盐水调整细胞数为2×10⁷ml^-1,取0.2ml肿瘤细胞于每只裸鼠左臀部消毒后皮下接种。（5）浸提液细胞悬液混合液的体内接种从生理盐水调整的细胞数为2×10⁷ml^-1的细胞悬液中取0.1 ml,再分别加入3种已配制好的骨水泥浸提液0.1ml,分别按高、中、低剂量注射于每只裸鼠右耳后、左耳后及右臀部。（6）大体观察每天观察裸鼠的活动、注射部位的皮肤和肿瘤生长情况。（7）抑瘤率计算于实验第12天,将各组裸鼠断头处死后,以无菌操作从背部皮下完整剥取肿瘤组织,剥离干净后于电子天平称瘤体质量并计算抑瘤率。（8）病理学观察称取裸鼠瘤体重量后,取出肿瘤块,固定、脱水、透明、石腊包埋后,切片机切片,烘干后脱蜡,常规HE染色。6统计学处理本研究所得的数据均为计量资料,数据用均数±标准差（（?）±s）表示,对一般计量资料选用重复测量的方差分析进行统计处理,组间多重比较用LSD检验;相同时间点的不同组间以及同组的不同时间点之间的比较,多组之间用单因素方差分析,两组之间比较用t检验。率的比较用非参数检验。将p＜0.05定义为有统计学意义,p＜0.01为有显著统计学意义。结果1载空白微球骨水泥的生物相容性（1）倒置显微镜下观察材料组和空白组的细胞均形态良好,生长旺盛。（2）细胞增殖测定CPC与CPC/M对细胞的增殖影响均不明显。随着培养时间的延长,细胞数量不断增加。加入CPC和CPC/M浸提液后,对细胞增殖有一定的抑制作用,但总体与空白组的细胞增殖曲线基本保持一致。（3）电镜观察在不含微球的骨水泥表面,仅有较少的细胞生长且细胞形态较差,细胞皱缩。而在载微球的骨水泥中,细胞数目较多且形态良好,细胞可在微球周围的骨水泥上生长,甚至有突触伸展至骨水泥表层的微球表面。（4）全身急性毒性试验分析各组小鼠在注射不同浸提液后的5天内的长势良好。并且未出现死亡或昏迷、呼吸抑制、呼吸困难、四肢活动受限等中毒反应。（5）溶血试验实验后肉眼下行大致观察,发现各实验组和阴性对照组无明显溶血,而阳性对照组溶血明显。CPC组和CPC/M组的比较接近,均低于阳性对照组,但高于阴性对照组;各组的溶血率均未超过5%。（6）微核试验对雌性动物和雄性动物分别进行卡方检验,雌、雄性动物的微核率均与阴性对照组无显著差异,与阴性对照组比较均有显著差异。（7）热原试验注射后6只家兔平均体温升高为0.06℃,不大于1.4℃。2包裹多柔比星微球骨水泥的制备和表征（1）扫描电镜观察PLGA微球的粒径在100～150μm之间,微球呈球形或椭球形,表面光整圆滑;骨水泥与药物混合后的微结构变化不大,都是由极小的微粒构成的,无法判断药物在骨水泥中的位置和特征性表现;载微球骨水泥的结构疏散,100～150μm大小的微球均匀分布于CPC粉末之间。（2）XRD分析3种样品的XRD谱线与标准的羟基磷灰石的XRD谱线一致,其主峰位于XRD谱线32°附近。加入药物和微球并没有新的相产生,主要转化产物仍是羟基磷灰石。（3）抗稀散性3种骨水泥刚投入生理盐水中材料均无崩解,但24小时后包裹微球的骨水泥表面有明显溃散,材料不完整。（4）骨水泥凝结时间CPC/M/D组凝结时间最长,CPC组凝结时间最短;37℃时,凝结时间较长;终凝时间较长,在CPC/M/D组可达45min左右。（5）可注射性加入药物微球的CPC/M/D组可注射性能最好,与CPC和CPC/D组比较,差异有显著统计学意义。而加入原药的CPC/D和CPC之间的可注射性能无显著差异。微球比原药更能提高骨水泥的可注射性能。（6）孔隙率CPC/M/D组的孔隙率最大,CPC组最小。CPC中加入药物微球后,其孔隙率可显著增加达61.67%,与CPC和CPC/D相比,差异均有统计学意义。（7）生物力学强度测试CPC组屈服应力最大,CPC/M/D组最小。当DOX原药和药物微球加入磷酸钙骨水泥后,其强度会有所降低,但两者之间差异并不显著。3包裹多柔比星微球骨水泥的体外释药和降解（1）微球的载药率和包封率3个样品的载药率和包封率均较低,分别为5.9±0.29（%）和74.1±3.57（%）,3个样本的载药率和包封率均比较接近。（2）微球的释药特性时间越长,累积释放的药物浓度越高,药物在微球内是缓慢释放的。药物释放过程仍存在一定程度的突释,以第1周释放最快,其后进入缓释状态。（3）含药物骨水泥的释药特性CPC/D释放药物较快,释放药物浓度很快到达并维持于最大值;CPC/M/D释放药物缓慢,释放药物逐渐减少,释放时间较长。CPC/M/D的释药量较低,而释药时间较长,表现出良好的缓释效果。（4）微球的降解空白微球早期的重量丢失较少,而4周后,重量丢失较多。DOX微球的重量丢失则在前2周比较迅速,而后期比较缓和。（5）骨水泥的重量丢失3种骨水泥均可以随时间延长重量丢失逐渐增大,载入微球以后的CPC/M/D在整个过程中重量丢失最快,但是在前2周,3种样本的重量丢失差距并不明显;而从第4周开始,CPC/M/D的重量丢失明显快于其它两种不含微球的样本。（6）骨水泥的强度CPC/D和CPC/M/D均在一定程度上降低了骨水泥的强度;前2周各组的强度差异很小,而后逐渐表现出显著差异。4包裹多柔比星PLGA微球骨水泥的体内成骨作用（1）大体观察结果术后各组动物进食及活动正常,切口无红肿、渗液等炎性反应,切口如期愈合。2周时,材料均大部分完整。4～8周时,3组材料己明显降解。16周时有少量新骨形成。（2）各组兔骨缺损区X射线检查结果16周时,3种植入体均逐渐与受体骨骨性愈合成一体,密度逐渐接近,骨皮质部分连接,材料逐渐降解,骨髓腔己通,以CPC/M/D的成骨最快,塑形最好。（3）生物力学测定测量结果CPC/MD/在前4周的强度比较弱,自第8周开始强度明显高于其它2组,此时3者的强度以CPC/M/D组最高,而CPC/D最低。（4）骨水泥在骨内组织相容性和成骨方式CPC和CPC/D组骨水泥在骨内是的分层降解的,而CPC/M/D是内外同时降解的。（5）骨水泥在骨内的降解速率CPC/M/D组骨水泥在术后16周时可见新形成的骨组织结构较正常的皮质骨疏松,骨水泥基本降解完全,正常骨皮质与新生骨小梁界面明显。（6）骨水泥及其降解物成分分析CPC/M/D组骨水泥术后3个月时剩余物中磷酸钙的含量显著下降,主要是钙盐的降解和成骨作用。5包裹载多柔比星PLGA微球骨水泥的抗肿瘤作用（1）倒置显微镜下观察空白组的肿瘤细胞在培养板中生长迅速。不同剂量组的包裹多柔比星微球骨水泥浸提液有一定抑制肿瘤生长的作用。但存活细胞均可进入增殖期,开始成倍繁殖。（2）SaoS-2细胞增殖测定各组浸提液对细胞增殖的抑制作用存在显著差异,以高剂量组最强,低剂量组最弱,说明浸提液对SaoS-2细胞有一定的抑制作用且在本研究的剂量范围内将随剂量的增大而增强。（3）裸鼠的大体表现对裸鼠种植肿瘤细胞后,前2天裸鼠略显倦态,活动减少,第3天开始逐渐恢复。一旦成瘤后,肿瘤迅速长大。（4）裸鼠瘤体质量及抑瘤率各浸提液组均有明显的抑制肿瘤生长作用。因此,肿瘤的抑制作用与剂量关系密切。高剂量组抑瘤率达61.0%,剂量越小,抑瘤率也越小。（5）肿瘤组织的病理学比较将获得的肿瘤组织块用HE染色后,在同等放大倍数下进行比较,大剂量的浸提液具有明显的抑瘤作用,可出现广泛的肿瘤坏死,肿瘤数量明显较少,而小剂量的浸提液抑瘤作用不明显,和模型组的肿瘤组织差异不明显。结论制得的包裹PLGA微球骨水泥具有良好的生物相容性,而复合药物PLGA微球的骨水泥同样具有良好的可注射性、力学强度可等一般特性,包裹多柔比星微球骨水泥具有良好的降解和药物缓释作用。当包裹多柔比星微球骨水泥植入体内填充骨缺损时,可以促进新骨形成的作用。包裹多柔比星骨水泥的浸提液在体外和体内都具有抗肿瘤作用。更多还原

【Abstract】 BackgroundCalcium phosphate cement （CPC） has been developed as an alternative bone substitute material because of its good biocompatibility, excellent osteo-conductibility and easy shaping in complicated geometries. However, the drawbacks of CPC, including its inferior mechanical strength, poor injectability and lack of macroporosity for bone ingrowth, have restricted its clinical applications. The macroporous （average diameter larger than 100 urn） cements can facilitate the ingrowth of new bone, which is able to enhance the mechanical strength in CPC. However, there is still a general conflict between macroporosity and mechanical strength in CPC at early stage. Therefore, it is very important to improve the mechanical strength in CPC before new bone ingrowth.The objective of the present study was to develop an injectable CPC containing poly（lactide-co-glycolide） （PLGA） microspheres. The biodegradable PLGA microspheres were used to impart in situ macroporosity to the cement. The novel cement can offer optimal strength at early stage and macroporosity for bone ingrowth after microspheres degradation. In addition, Doxorubicin microspheres, instead of control microspheres, will be incorporated with CPC to obtain a drug delivery system. Further investigation will be made on the osteogenesis andantitumor effect of the composite.ObjectiveThe aim of the present study is to investigate the general properties, osteogenesis and antitumor effect of the novel CPC containing Doxorubicin PLGA microspheres. The ultimate purpose of the present study is to discuss the possibility of clinical application of the novel cement.Methods1 Biocompatibility of the cement incorporated with PLGA microspheres （1） Preparation of CPC powder: The CPC powder used in this study was prepared by mixing partially crystallized calcium phosphate （PCCP） and dicalcium phosphate anhydrous （DCPA） at a mass ratio of 1:1. （2） Preparation of PLGA microspheres: PLGA microspheres were made by a solvent evaporation method. （3） Preparation of control cement samples: sodium citrate solution was Sodium citrate solutions with concentrations of 15 wt.% were used as the liquid phase in this study. Then the CPC powder was homogeneously mixed with sodium citrate solution at a liquid to CPC ratio of 0.4 ml g^-1. The cements with a pillar shape were made in a special mould.（4） Preparation of cement samples with PLGA microspheres: The PLGA microspheres were uniformly mixed with CPC powder at a PLGA to CPC weight ratio of 30/70. The cement samples incorporated with PLGA microspheres were thus made.（5） Grouping methods: Based on the difference of materials, 3 groups determined as Control Group, CPC Group and CPC/M Group were used for cytocompatibility study, and the latter two groups were divided into three subgroups respectively by different concentration or dose for in vivo biocompatibility.（6） Preparation for extracts: Samples were weighed and sterilized, then immersed into cell culture fluid at a weight volume ratio of 1g:10ml. Leaching liquid was obtained after the samples being immersed for 72h at 37℃, with 5 vol% CO₂.（7） Cell proliferation curve: Human bone marrow stem cells （hBMSCs） suspension was seeded in 96 hole plate at a volume of 200μL per hole, cells then were incubated for 24 hours at 37℃in a incubator with 5 vol% CO₂ . Cell culture fluid was poured, instead, isovolumic extracts were added into the holes. Cells were incubated again for 2, 4, 6, 8, 10 days respectively. Cell Counting Kit-8 was added into the holes, OD value of cells in each hole was measured under the wave length of 450 nm.（8） Observe cells under optical microscope and electron microscope: Cells incubated in different medium were observed under optical microscope and taken pictures. HBMSCs were seeded onto plaster samples and then observed under electron microscope.（9） Acute toxicity testing: 30 mice was divided into three groups as normal sodium （NS） Group, CPC Group and CPC/M Group, 5 female and 5 male mice was selected to each group, 1ml solution in each group was injected into mice by intratailvenous injection. The weight and behavior of each mouse were recorded 24h, 48h, 72h and 120h after injection.（10） Hemolysis test: Fresh human anticoagulated blood was diluted into suspension at a volume concentration of 2%. Cement samples were immersed into NS to regulate the concentration of suspension to 0.1 g·ml^-1, 0.05g·ml^-1 and 0.01g·ml^-1. NS was used as negative group while double distilled water as positive group. 2 ml different liquor was mixed with 2 ml human blood and then accepted water bath at 37℃for 1 hour, centrifugalization at 1000 r·min^-1 for 5 minutes. 4 ml 0.1vol% Na₂CO₃ solution was added into 1 ml supernatant fluid extracted from suspensions. OD value of was measured under the wave length of 540 nm and hemolytic ratio was then calculated by accordant values.（11） Micronucleus test: 80 healthy mice with half male and half female were randomly divided into eight groups: 60 mice in 6 experimental groups were injected in different doses （1, 2, 4 ml） of the material saline extracts, the other two groups were regarded as a negative control group （NS） and a positive control group (cyclophosphamide 40 mg·kg^-1). All the animals were killed and bone marrow was extracted and mixed with calf serum, Rett’s （Wright） staining was performed later. In the oil microscope count, micro-nucleus cells of 1000 polychromatic erythrocytes in each animal were recorded. The total micro-nucleus cells of 5000 polychromatic erythrocytes were calculated as the micro-nucleus ratio.（12） Pyrogen test: 6 healthy rabbits were selected and 5 ml suspensions of different samples were injected into each animal. The temperature of each rabbit was measured 3 times before and after injection, the difference was recorded as increased temperature.2 Characterization and preparation of calcium phosphate cements containing Doxorubicin PLGA microspheres（1） Grouping methods: Based on the difference of materials, 3 groups determined as Control Group, CPC/D Group and CPC/M/D Group.（2） Observation by eletron microscope: Surface structure of different samples was observed by eletron microscope. The average diameter of microspheres was measured by their images.（3）X-ray diffraction （XRD） analysis: Powder X-ray diffraction （XRD） analysis was used to estimate the extent of CPC and CPC/M/D samples.（4）Washout Resistance Test: shape the samples into spheres by hand, then put them immediately into NS or distilled water （37℃） under slightly shock for 24 h. The samples were considered to show good property of washout resistance if it did not visibly disintegrate in the liquor.（5） Setting time measurement: The initial and final setting time of cement samples in 3 different groups were measured at 25℃and 37℃respectively. 6 samples from each group were selected and measured. The average values of temperature were recorded.（6） Injectability: The injectability of different samples was tested with a syringe of 14.5 mm inner diameter, which was fitted with a needle of 1.6 mm inner diameter. After mixing different powder with sodium citrate solution of 15 wt.% for 1 min, the as-prepared paste was poured into the syringe. A 5 kg compressive load was then mounted vertically on the top of the plunger for 2 min. The injectability was calculated according to the percentage of mass expelled from the syringe to total mass before injection.（7） Interval porosity: Interval porosity was measured by dewatering method. The volume of samples and pores in samples were calculated. The volume ratio of pores to samples was recorded as interval porosity.（8） Maxium strength of compression （MSC）: The compressive strength of the specimens was measured using a universal material testing machine at a crosshead speed of 0.5mm·min^-1.3 In vitro drug release and degradation of calcium phosphate cement containing Doxorubicin PLGA microspheres（1） Drug load and encapsulation of Doxorubicin microspheres: Doxorubicin loading in microspheres was determined using a UV-visible spectrophotometer. Freeze-dried microspheres （5mg） were dispersed in dimethyl sulfoxide （DMSO）. The concentrations of gentamicin releasing in vitro was calculated referring to the standard curve of realationship between the concentrations of Doxorubicin and their OD values. The experiment was repeated three times. （2） In vitro drug release from Doxorubicin microspheres: 48 bottles of microspheres with each weight of 100 mg were added into simulated body fluid and immersed for 1, 3, 5, 7, 10, 14, 21, 28 days respectively. Supernatants from 6 portions randomly selected each time were measured at 480 nm by UV-visible spectrophotometer.（3） In vitro drug release from different plasters: 2 different cement samples were added into simulated body fluid and immersed for 1, 3, 5, 7, 10, 14, 21, 28 days respectively. Supernatants from fluid at each interval were collected and measured at 480 nm by UV-visible spectrophotometer.（4） In vitro degradation of microspheres: 30 bottles of microspheres with each weight of 100 mg were added into simulated body fluid and immersed for 7, 14, 28, 42 and 56 days respectively. 6 portions were randomly selected each time, the supernatants were pored. Microspheres were weighed after being dried in vacuum for 2 days. The difference between the weight of microspheres before and after immersion was regarded as weight loss of microspheres.（5） In vitro degradation of plasters: 35 samples were selected and weighed before immersion in simulated body fluid for 7, 14, 28, 42 and 56 days respectively. 6 portions were randomly selected each time, the supernatants were pored. Samples were weighed after being dried in vacuum for 2 days. The difference between the weight of cement samples before and after immersion was regarded as weight loss of cement samples.4 Osteogenesis of calcium phosphate cement containing Doxorubicin PLGA microspheres（1） Animals and grouping method: 40 rabbits weighed from 400 to 450 gram were randomly divided into 20 pairs. The radius of rabbits were divided into four groups: Control group, CPC group, CPC/D Group and CPC/M/D group. （2） Preparation for animal models: Intravenous anesthesia was performed before radius being exposed. Radius defect with a length of 1.5cm was made bilaterally. Different samples were implanted into the defective bone.（3） General observation: Observe the appetite, behavior, sound situation and investigate the degradation of implants, osteogenesis of samples postoperatively.（4） X-ray examination: 5 pairs of rabbits were selected each time at 2, 4, 8 and 16 weeks postoperatively. X-ray examination was performed to learn bone growth and degradation of samples.（5） Mechanical strength: The samples of radius were obtained to measure mechanic strength after 5 rabbits being killed each 2, 4, 8 and 16 weeks after operation.（6） Residual calcium and phosphate in cement samples: XRD were performed at the 4^th postoperative week to analyze the residual calcium and phosphate in cement samples.（7） Pathology of radius samples: Each radius was obtained after killing the rabbits 16 weeks after operation. The samples were fixed, embedded and sliced successively. Pathology of samples was observed in optical microspheres after hematoxylin-eosin staining.5 Antitumor effect of calcium phosphate cement incorporated with Doxorubicin PLGA microspheres（1） SaoS-2 cells culture: SaoS-2 cells were transferred to centrifuge tube to accept centrifugalization for 10min. Supernatant was poured and RPMI - 1640 nutritive medium containing 10 Ml 10vol% calf serum was added. Centrifuge for another 10 minutes and pour the supernatant again, add moderate nutritive medium into culture flask after cells being transferred. Cells were incubated at 37℃in a incubator with 5 vol% CO₂. （2） Preparation for extracts: Samples were weighed and sterilized, then immersed into cell culture fluid at a weight volume ratio of 1g:20ml, 1g:10ml and 1g:5ml. Leaching liquid was obtained after the samples being immersed for 72h at 37℃, with 5 vol% CO₂.（3） SaoS-2 cells inhibition: SaoS-2 cells suspension was seeded in 96 hole plate at a volume of 200μL per hole, cells then were incubated for 24 hours at 37℃in a incubator with 5 vol% CO₂ . Cell culture fluid was poured, instead, isovolumic extracts were added into the holes. Cells were incubated again for 5 days. Cell Counting Kit-8 was added into the holes, OD value of cells in each hole was measured under the wave length of 490 nm.（4） Preparation of osteosarcoma models: Regulate the amount of SaoS-2 cells to 2×10⁷ml^-1 by the addition of NS. 0.2ml cells were injected into left buttock of each nude mouse.（5） Injection of cells with materials: Regulate the amount of SaoS-2 cells to 2 X 10⁷ml^-1 by the addition of NS. 0.1ml cells with 0.1ml cement supernatant with different concentration were injected into right buttock and both post aurems of each nude mouse.（6） General observation: Observe the appetite, behavior, injection area and growth of tumors postoperatively.（7） Tumor inhibition: Kill nude mice 12 days after cells injection, extract the total tumor tissues and weigh them one by one. Calculate the ratio of tumor inhibition by correspondent weight.（8） Pathology of tumor: Each tumor was obtained after killing the mice postoperatively. The samples were fixed, embedded and sliced successively. Pathology of samples was observed in optical microspheres after hematoxylin-eosin staining. 6 Statistical analysisThe software package SPSS 17.0 was applied for statistical analysis, including t test, x² test, one-way ANOVA method of square-deviation （SD）.Results1 Biocompatibility of the cement incorporated with PLGA microspheres（1） Observation in inverted microscope: Cells grew well in control group and materials groups with good activity and shape.（2） Cell proliferation: Calcium phosphate cement and the cement incorporated with microspheres nearly have no influence on the proliferation of cells.（3） Observation by electron microscope: Little cells could grow on the surface of solo-calcium phosphate cement and shrinkage of cells could be seen. Many cells could be seen growing well on the surface of calcium phosphate cement containing microspheres.（4） Acute toxicity test: The increase of weight in different materials groups was the same as that in NS group. Mice were all active without abnormal behavior.（5） Hemolysis test: Hemolysis did not appear in negative control group and different cement groups, but could be found clearly in positive control group. The difference between cement groups and negative control group was not significant, but the difference was significant compared to positive control group. The hemolysis ratio was less than 5% in each cement group.（6） Micronucleus test: The number of micronucleus in each cement group was less than 8‰. Compared with negative control group, no significant difference appeared in cement groups. However, the difference between cement groups and positive control group was significant.（7） Pyrogen test: Rectal temperature of rabbits increased a little after injection. the average increased temperature was 0.06℃, lower than 1.4℃. 2 Characterization and preparation of calcium phosphate cements containing Doxorubicin PLGA microspheres（1） Observation by scanning electron microscope: The average diameter of microspheres was between 100 and 150μm. Microspheres were globular and the surface was slick and sly. Calcium phosphate cement was made up of microparticles, the drug could not be found when mixed with cement powers. Microspheres with the average diameter between 100 and 150μm could be observed evenly adhering to calcium phosphate cement microparticles.（2） X-ray diffraction: All the X-ray diffraction pattern of three different sorts of cement revealed characteristic peaks of nano-HA and no secondary phases.（3） Washout resistance: Solo- calcium phosphate cement showed excellent washout resistance. It remained stable while immersed in NS, no obvious collapse phenomenon was observed. However, obvious collapsing appeared in both other two samples.（4） Setting time: The results showed that the addition of microspheres can significantly prolong the composite bone cement setting time, including both the initial and final time. The final setting time of calcium phosphate cement containg microspheres could delay to 45 minutes.（5） Injectability: The calcium phosphate cement incoporated with Doxrorubicin microspheres showed best property of injectability among the three kinds of cement.（6） Interval porosity: Interval porosity was up to 61.67%, which was the most in the calcium phosphate cement incoporated with Doxrorubicin microspheres.（7） Mechanical strength: The strength of calcium phosphate cement dramatically decreased while Doxrorubicin microspheres being incoporated. The decrease was not significant compared with the cement mixed with Doxorubicin powder.3 In vitro drug release and degradation of calcium phosphate cement containing Doxorubicin PLGA microspheres（1） Drug loading and encapsulation: Drug loading and encapsulation were 5.9±0.29（%） and 74.1±3.57（%） respectively.（2） Drug release from microspheres: Drug in microspheres released fast at the first week but lowered later.（3） Drug release from cement samples: Drug mixed with CPC powder directly released faster than that in the CPC incorporated with Doxorubicin microspheres. CPC containing Doxorubicin microspheres showed good property of slow-release.（4） Degradation of microspheres: Weight loss of control microspheres was less at the early stage than that after 4 weeks. The weight loss of Doxorubicin microspheres was fast at the first two weeks but became slow since then.（5） Degradation of cement samples: The strength of CPC decreased fastest wile Doxorubicin microspheres incorporated, and the acceleration of decrease began to be much faster than the other two samples since 4 weeks on.（6） Compressive strength of cement samples: The difference between 3 kinds of samples was not significant at the first 2 weeks. However, the difference became more and more significant between the cement containing Doxorubicin microspheres and the solo-cement since they were immersed for 4 weeks.4 Osteogenesis of calcium phosphate cement with Doxorubicin PLGA microspheres（1） General observation: Inflammatory reaction was not obvious in different materials groups and the skins were seemed the same as those in NS group. Mice were all active without abnormal behavior. All incisions cured as scheduled. CPC containing Doxorubicin microspheres degraded fastest in the three materials groups and new bone could be observed growing into the cement samples after implantation for 16 weeks. （2） X-ray examination: The shape of CPC containing Doxorubicin microspheres began to change at the 4^th postoperative week, and the residual cement containing Doxorubicin microspheres disappeared after 16 weeks.（3） Mechanical strength: The strength of defected bone increased gradually after cement samples implantation. The strength of bones reconstructed by CPC containing Doxorubicin microspheres increased fast after implantation for 8 weeks.（4） Osteogenesis of cement samples: The cement containing Doxorubicin microspheres combined to bone compactly. New bone could be seen in the outer layer of CPC and CPC/Doxorubicin composites, but the ingrowth of new bone could be found in the center of CPC containing Doxorubicin microspheres.（5） Acceleration of cement degradation: CPC containing Doxorubicin microspheres degraded fastest among three sorts of cement samples. Degradation only could be observed at the bone-bone cement interface after implantation for 16 weeks, however, the new bone has grown into the CPC containing Doxorubicin microspheres.（6） Component analysis of residual cement samples: Content of calcium phosphate decreased dramatically at the 3rd month after implantation of CPC containing Doxorubicin microspheres, which indicated that the degradation of calcium salts played an important role in the degradation of CPC containing microspheres.5 Antitumor effect of calcium phosphate cement incorporated with Doxorubicin PLGA microspheres（1） Observe cells under optical microscope: Cells grew fast in control group and CPC group. Dead cells could be seen in CPC/M/D groups at the first two days after incubation, but the survival cells began to recover and grew fast two days later.（2） Cells proliferation: Inhibition of cells proliferation appeared in CPC/M/D groups after incubation in the extracts of CPC/M/D. The OD values in high dose group were lower than those in low dose group, which indicated that the inhibition effect was related to its dose.（3） General observation on mice: Nude mice seemed to be tired at the first two days after tumor cells injection. They became active after 3 days. Tumor could be observed growing fast after about 7 days.（4） Weight of tumor and tumor inhibition rate: The higher dose of CPC/M/D extract was injected, the smaller the tumor formed, which suggested that the tumor inhibition effect was positively related to its dose. The inhibition rate of high dose group could reach to 61.0%.（5） Pathology of tumor: Tumor necrosis could be found in high dose group, but nearly no necrosis appeared in low dose group.ConclusionsCPC containing PLGA microspheres exhibited good biocompatibility. When Doxorubicin microspheres were incorporated into CPC, the resultant production also showed good properties such as compressive strength, injectability, etc. The novel cement could degrade and release drug slowly in vitro. When the composites were implanted into defected bone, it could facilitate the ingrowth of new bone. The extracts of CPC containing Doxorubicin also could inhibit the Cells proliferation of tumor both in vitro and in vivo.更多还原